Nucleic acids are used frequently in the clinical setting to identify genetic mutations and to diagnose bacterial and/or viral infections. Such methods generally require use of nucleic acids isolated or extracted from biological samples.
A commonly used method for isolating DNA from a biological sample (e.g., blood or saliva) involves lysing the cells contained in the sample with a combination of a proteolytic enzyme and a detergent followed by extracting the mixture with an organic solvent, e.g., phenol and chloroform, so that the DNA enters the aqueous phase and the protein enters the organic phase. The DNA in the aqueous phase is then concentrated by alcohol precipitation and re-suspended in a suitable volume for analysis. Such methods are, however, time-consuming and generally require the use of toxic reagents.
Simpler methods using fewer reagents have been reported. For example, DNAzol™ Direct (Molecular Research Center, Inc.) is an alkaline solution containing polyethylene glycol and other additives. Buccal swabs and whole saliva samples are incubated in DNAzol™ Direct for 15 min. The resulting lysate may be added directly to a polymerase chain reaction (PCR) mix, where the lysate should be less than 10% of the PCR mix by volume.
In another example, 0.25 μl to 5 μl of whole saliva or buccal swab sample may be added directly into a 50 μl PCR mixture containing 1× EzWay™ Direct PCR Buffer (Koma Biotech). The PCR cycling program should use an initial denaturation of 95° C. for 5 min followed by cycling denaturation steps of 94° C. for 30 sec to 60 sec.
The disadvantages of DNAzol™ Direct and EzWay™ Direct PCR Buffer are that they require the use of special reagents and specific incubation or denaturation conditions.
Another approach to bypassing the DNA purification step is the use of specially-modified DNA polymerase enzymes that are resistant to inhibitors in samples. For example, the Phusion™ Human Specimen Direct PCR Kit (Finnzymes) claims to allow PCR directly from unpurified human samples. A tiny amount of sample is used directly in the PCR reaction with no prior purification steps. Suitable sample materials include buccal swabs, saliva, amniotic fluid, hair, fingernails, teeth, and skin biopsies. The kit is based on modified Phusion™ Hot Start II High-Fidelity DNA Polymerase. This specially engineered proofreading DNA polymerase is fast, robust and highly tolerant of many PCR inhibitors present in human tissues. The disadvantage of this approach is that it requires a proprietary DNA polymerase that may be more expensive than standard DNA polymerase.
Simpler methods for analyzing whole saliva samples have been reported. For example, Ochert et al. (1994) boiled whole saliva for 5 min and then used it as the sample for PCR. This method resulted in successful PCR for 7 out of 10 samples. The researchers used 30 IA of boiled sample in a total reaction volume of 50 μl. The PCR cycling program used cycling denaturation steps of 1 min at 94° C. One of the samples, which gave an inconclusive result with 5 min of boiling, generated a positive result after 6 or more minutes of boiling. The researchers concluded that: “[t]he chemical nature of the inhibitors remains to be characterized. By inference, they are unlikely to be proteins because of their relative heat resistance and the observation that extraction procedures based on phenol-chloroform or proteinase K did not prevent inhibition. The persistence of inhibition after processing with gel matrices or ion-exchange resins suggests that low-molecular-weight ionic moieties are also not inhibitors. Polysaccharides are possible candidates.” (Ochert A S et al. (1994) Inhibitory effect of salivary fluids on PCR: potency and removal. Genome Research. 3: 365-368.)
In another example, French et al. (2002) performed direct PCR amplification from whole saliva using HyBeacon™ probes on the LightCycler™ instrument. PCR volumes were typically 20 μl, containing 2 μl of saliva. Saliva samples were diluted to 50% in water. The PCR mixtures used 1 unit of Taq DNA polymerase (Taq from Amersham Pharmacia Biotech or Z-Taq from TaKaRa). The final concentration of PCR primers was 0.5 μM each, and the final concentration of HyBeacon™ fluorescent probe was 150 nM. The PCR cycling program used an initial denaturation step of 95° C. for 5 min followed by cycling denaturation steps of 95° C. for 0 seconds (i.e., the thermal cycler ramped up to 95° C. and immediately ramped down to the extension temperature). Saliva samples were typed on the same day that they were collected, although a significant reduction in assay efficiency was not observed with saliva samples stored at 4° C. for 2-3 days, or −20° C. The researchers noted that since the number of buccal epithelial cells shed into saliva samples may not be equal from person-to-person and day-to-day, a further study of assay reproducibility and robustness may be required. (French D J et al. (2002). Ultra-rapid DNA analysis using HyBeacon™ probes and direct PCR amplification from saliva. Molecular and Cellular Probes. 16: 319-326.)
Researchers have found that there is high variability in the concentration of DNA from saliva or buccal swabs in a population of subjects. For example, Garcia-Closas et al. (2001) collected two cytobrush samples each from 40 individuals and found that the amount of human DNA ranged from 0.006 to 13.5 μg after phenol-chloroform DNA purification (Garcia-Closas M et al. (2001). Collection of Genomic DNA from Adults in Epidemiological Studies by Buccal Cytobrush and Mouthwash. Cancer Epidemiology, Biomarkers & Prevention. 10(6): 687-696.).
Although boiling whole saliva for 5 minutes or more, or diluting whole saliva and then denaturing at 95° C. for 5 min are relatively simple steps, they still require time and effort on the part of the user. For automated instruments, these steps would require extra mechanical parts and movements to accomplish. In addition, the PCR success rate of 7 out of 10 boiled saliva samples reported by Ochert et al. (1994) makes this method less desirable for clinical applications where higher reliability is typically expected.